Most biological processes involve specific protein-protein interactions (PPIs). General methodologies to identify interacting proteins or to study these interactions have been extensively developed. Among them, the yeast two-hybrid system currently represents the most powerful in vivo approach to screen for polypeptides that could bind to a given target protein. As well known, this system utilizes hybrid genes to detect PPIs by means of direct activation of a reporter-gene expression. In essence, the two putative protein partners are genetically fused to the DNA-binding domain of a transcription factor and to a transcriptional activation domain, respectively. A productive interaction between the two proteins of interest will bring the transcriptional activation domain in the proximity of the DNA-binding domain and will trigger directly the transcription of an adjacent reporter gene (usually lacZ or a nutritional marker) giving a screenable phenotype. The transcription can indeed be activated through the use of two functional domains of a transcription factor: a domain that recognizes and binds to a specific site on the DNA and a domain that is necessary for activation.
Other approaches have been proposed to monitor PPIs in intact eukaryotic cells (e.g., using a mammalian “two-hybrid” system based on β-galactosidase complementation), or to screen complex libraries of proteins for direct interaction with a given ligand (e.g., phage display and double-tagging assay). However, these techniques do not allow an in vivo selection of the relevant clones.
The present inventors previously described an assay (called “BACTH” for “Bacterial Adenylate Cyclase Two-Hybrid”) that uses Escherichia coli as a host and involves the interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade (Karimova G. et al, PNAS, 1998, 95(10):5752-5756). In this system, the proteins of interest were genetically fused to two complementary fragments, T25 and T18, from the catalytic domain of Bordetella pertussis adenylate cyclase (AC), and co-expressed in an E. coli Δcya strain (i.e., deficient in its endogenous adenylate cyclase). Association of the two hybrid proteins resulted in functional complementation between the separately inactive T25 and T18 fragments leading to cyclic AMP (cAMP) synthesis. In E. coli, cAMP binds to the catabolite activator protein (CAP or CRP) and triggers the transcriptional activation of catabolic operons, such as lactose or maltose, thus yielding a characteristic phenotype. This system has been extensively used to reveal a wide variety of interactions between bacterial, eukaryotic, or viral proteins, occurring at various subcellular locations, e.g. cytosol, membrane or DNA level (Karimova G. et al, J. Bacteriol 2005, 187(7):2233-2243; Karimova G. et al, J. Bacteriol. 2009, 191(1):333-346; Karimova G. et al, J. Mol. Microbiol. Biotechnol. 2001; 3(1):73-82).
This BACTH system is however not adapted for specific applications for the following reasons.
In the absence of its activator calmodulin (CaM), AC exhibits a kcat of about 1-2 s−1, and therefore few hundreds of active hybrid protein complexes per bacteria are required to produce enough cAMP to confer a cya+ phenotype to the E. coli Δcya host cell. Consequently, few hundreds of chimeric molecules have to be expressed in each bacterium. Given that the volume of a bacterium is very small (typically within the femtolitre range), the probability that proteins of interest interact is relatively high, due to spatial constraints. A background level of response thus lowers the sensitivity of the BACTH system, resulting in an increased number of false positives. Indeed, these spatial constraints (numerous molecules in a small area) facilitate interactions between two proteins even when their affinity is medium or low. Thus, the BACTH system does not enable to discriminate protein interactions on the basis of the affinity level of the proteins. This means that one will not be able to conclude, using the BACTH system, if the protein interactions are of low, medium, or high affinity.
Moreover, as the moiety of interest is expressed at high level in the bacterial cells, the BACTH system cannot be used to test interactions involving toxic proteins, that may impair the host cell metabolism and thus interfere with the cAMP-related signaling cascade.
Therefore, there is a need in the art of a more sensitive system for detecting high-affinity protein interactions, e.g., those involving integral membrane proteins, antigen/antibody (or recombinant single-chain antibody fragment, scFv, or other protein scaffold displaying affinity for a given target protein), or toxic proteins.